Superscript first strand synthesis system for rt pcr

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, Germany, United Kingdom

The SuperScript First-Strand Synthesis System for RT-PCR is a kit designed for the reverse transcription of RNA into cDNA. It provides the necessary components for the first-strand cDNA synthesis step, which is a critical part of the reverse transcription-polymerase chain reaction (RT-PCR) process.

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First-Strand Synthesis System (55 citations) RT-PCR system (41 citations) SuperScript II First-Strand Synthesis System for RT-PCR (35 citations) SuperScript RT-PCR system (20 citations) SuperScript III First-Strand cDNA synthesis system for RT-PCR (6 citations) SuperScript First-Strand System for RT-PCR (5 citations) Superscript First-Strand cDNA Synthesis system for RT-PCR (5 citations) Superscript First Strand RT-PCR system (3 citations) SuperScript IV First-Strand Synthesis System for RT-PCR Kit (3 citations) Superscript III First-Strand Synthesis System for real-time PCR (RT–PCR) (2 citations)

305 protocols using superscript first strand synthesis system for rt pcr

Isolation of Total RNA and cDNA from A. suum

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Twenty-thirty A. suum heads were flash-frozen in liquid nitrogen and ground to a fine powder. Total RNA was isolated using a Nucleospin Nucleic Acid Purification Kit (Clontech, Mountain View, CA). First strand cDNA was generated for PCR using a Superscript First Strand Synthesis System for RT-PCR (Life Technologies, Grand Island, NY). Rapid Amplification of cDNA Ends (RACE)-ready cDNA for 5′- and 3′-RACE was created from total RNA using a SMARTer RACE cDNA amplification kit (Clontech) according to the manufacturer’s instructions.

Konop C.J., Knickelbine J.J., Sygulla M.S., Vestling M.M, & Stretton A.O. (2015). Different Neuropeptides are Expressed in Different Functional Subsets of Cholinergic Excitatory Motorneurons in the Nematode Ascaris suum. ACS chemical neuroscience, 6(6), 855-870.

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First-strand cDNA Synthesis using SuperScript

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First-strand cDNA synthesis was carried out by the SuperScript First-Strand Synthesis System for RT-PCR (Life technologies, USA) according to manufacturer’s protocol, using 7μl of isolated total RNA as starting template.

Economopoulou P., Koutsodontis G., Avgeris M., Strati A., Kroupis C., Pateras I., Kirodimos E., Giotakis E., Kotsantis I., Maragoudakis P., Gorgoulis V., Scorilas A., Lianidou E, & Psyrri A. (2019). HPV16 E6/E7 expression in circulating tumor cells in oropharyngeal squamous cell cancers: A pilot study. PLoS ONE, 14(5), e0215984.

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Quantification of Exosomal miRNAs and mRNA

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The exosomal miRNAs were detected by qRT-PCR as described previously [37 (link)]. MiDETECT A Track™ miRNA qRT-PCR kits containing miRNA-specific forward primers and sequence complementary to the poly (T) adapter as the reverse primer were purchased from Riobio (Riobio Inc, Guangzhou, China). According to the manufacturer's instructions, total exosomal RNA from 250 ul mouse serum was polyadenylated and reverse-transcribed into cDNA using a poly (T) adapter. Mature miRNAs expression was detected using the SYBR^® Premix Ex Taq™ II, Perfect Real Time kit (TaKaRa, Dalian, China) on a CFX96 PCR system (Bio-Rad; Hercules, CA, USA). The relative expression of the detected miRNAs was expressed as ΔCT = 35- mean CTmiRNA.
Total RNA was extracted from RAW264.7 cells using Trizol (Life Technologies, Carlsbad, CA). RNA quantity and quality were determined by a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). One microgram of total RNA was used to synthesize the cDNA via the Superscript First-Strand Synthesis System for RT-PCR (Life Technologies, Carlsbad, CA). Primer sequences used for subsequent QPCR analysis were provided in Supplementary Table 1. The mRNA levels were normalized against β-actin expression and the relative expression level of each gene was presented as 2^−ΔΔCt.

Zhou X., Jiao Z., Ji J., Li S., Huang X., Lu X., Zhao H., Peng J., Chen X., Ji Q, & Ji Y. (2017). Characterization of mouse serum exosomal small RNA content: The origins and their roles in modulating inflammatory response. Oncotarget, 8(26), 42712-42727.

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Comprehensive Gene Expression Profiling

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Total RNA was isolated from cells using RNeasy Mini kit (QIAGEN). Reverse transcription polymerase chain reaction (RT-PCR) was performed using SuperScript First Strand Synthesis System for RT-PCR (Life Technologies). Real-time PCR was performed using SYBR Green system on a Light Cycler 480 instrument (Roche Diagnostics). Target gene expression was calculated by the ΔΔCt method and normalized to 18S ribosomal RNA expression levels. General PCR performed using PrimeSTAR Max DNA polymerase (Clontech). The following primer sequences were used: MCL1 forward; 5′-GAGGGCGACTTTTGGCTAC-3;, reverse; 5;-GTACCCGTCCAGCTCCTCTT-3;, FGFR1 forward; 5′-TGAAGATGATCGGGAAGCAT-3′, reverse; 5;-GGCCGTTGGTTGTCTTTTT-3;, FGFR2 forward; 5′-GATGGTGCGGAAGATTTTGT-3′, reverse; 5′-CCAGGTGGTACGTGTGATTG-3′, FGFR3 forward; 5′-TGGGCTTCTTCCTGTTCATC-3′, reverse; 5′-TCAGTGGCATCGTCTTTCAG-3′, FGFR4 forward; 5′-GTGCTGGTGACTGAGGACAA-3′, reverse; 5′-AGACAGAATCGCTGGAGGAG-3′, CK-19 forward; 5′-TTTGTGTCCTCGTCCTCCTC-3′, reverse; 5′-AGAGCCTGTTCCGTCTCAAA-3′

Kabashima A., Hirsova P., Bronk S.F., Hernandez M.C., Truty M.J., Rizvi S., Kaufmann S.H, & Gores G.J. (2018). Fibroblast Growth Factor Receptor Inhibition induces loss of matrix MCL1 and necrosis in Cholangiocarcinoma. Journal of hepatology, 68(6), 1228-1238.

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Rapid cDNA Amplification from A. suum Heads

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Twenty-thirty A. suum heads were flash-frozen in liquid nitrogen and ground to a fine powder. Total RNA was isolated using a Nucleospin Nucleic Acid Purification Kit (Clontech, Mountain View, CA, USA). First strand cDNA was generated for PCR using a Superscript First Strand Synthesis System for RT-PCR (Life Technologies, Grand Island, NY, USA). Rapid amplification of cDNA ends (RACE)-ready cDNA for 5′- and 3′-RACE was created from total RNA using a SMARTer RACE cDNA amplification kit (Clontech) according to the manufacturer’s instructions.

Konop C.J., Knickelbine J.J., Sygulla M.S., Wruck C.D., Vestling M.M, & Stretton A.O. (2015). Mass Spectrometry of Single GABAergic Somatic Motorneurons Identifies a Novel Inhibitory Peptide, As-NLP-22, in the Nematode Ascaris suum. Journal of the American Society for Mass Spectrometry, 26(12), 2009-2023.

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Quantifying Gene Expression via qRT-PCR

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Reverse transcription was carried out using the SuperScript First‐Strand Synthesis System for RT‐PCR (Life Technologies; Thermo Fisher Scientific, Waltham, MA, USA) or ReverTra Ace (Toyobo, Osaka, Japan) following the manufacturer's instructions. Total RNA (0.2–1.0 μg) was used for RT. Reverse‐transcribed cDNAs were subjected to real‐time PCR, which was carried out with a CFX96 Touch Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA, USA). For the detection of FUCA1, PHLDA3, and GAPDH, custom‐designed TaqMan Dual‐labeled Probes from Applied Biosystems (Foster City, CA, USA) (FUCA1: Hs00609173_m1) or from Sigma‐Aldrich (St. Louis, MO, USA) (PHLDA3 and GAPDH) were used. Data are shown as the mean fold expression ±SD.

Ezawa I., Sawai Y., Kawase T., Okabe A., Tsutsumi S., Ichikawa H., Kobayashi Y., Tashiro F., Namiki H., Kondo T., Semba K., Aburatani H., Taya Y., Nakagama H, & Ohki R. (2016). Novel p53 target gene FUCA1 encodes a fucosidase and regulates growth and survival of cancer cells. Cancer Science, 107(6), 734-745.

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RNA Extraction and RT-PCR Analysis

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RNA from tissue samples were extracted with the RNeasy kit (Qiagen) and reverse-transcribed to cDNA using SuperScript First-Strand Synthesis System for RT-PCR (Life Technologies). TaqMan Universal PCR Master Mix (Life Technologies) was mixed with 200 ng of cDNA, and the reactions were conducted using 7500 Real-Timer PCR System (Life Technologies). GAPDH was used as an endogenous control. All the primers for RT-PCR reactions were listed in Table 1. The data were analyzed with the 7500 Software. The RT-PCR was performed in triplicates.

Xu Z., Wang Y., Zhang L, & Huang L. (2014). Nanoparticle-Delivered Transforming Growth Factor-β siRNA Enhances Vaccination against Advanced Melanoma by Modifying Tumor Microenvironment. ACS Nano, 8(4), 3636-3645.

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iPSC RNA Extraction and qPCR Analysis

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RNA from iPSCs was extracted using the RNase Mini kit (Qiagen, CA). One microgram of total RNA was converted to complementary DNA using SuperScript First-Strand Synthesis System for RT-PCR (Life Technologies, CA). Real time PCR was performed using a GeneAmp 7900 sequence detection system with POWER SYBR Green (Applied Biosystems, CA). Gene expression of GAPDH levels was used as a loading control. Real time PCR data were presented after normalization with GAPDH expression. Primers used in the current study are listed in Table 1.

Zhao H.H, & Haddad G.G. (2022). Alzheimer’s disease like neuropathology in Down syndrome cortical organoids. Frontiers in Cellular Neuroscience, 16, 1050432.

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Liver RNA Extraction and RT-qPCR

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RNA from liver tissues was extracted using Trizol reagent (Life Technologies) following the protocol provided by the manufacturer. The first strand cDNA was synthesized from 250 ng mRNA using the SuperScript First-Strand Synthesis System for RT-PCR (Life Technologies). Reactions with (RT+) and without (RT-) the reverse transcriptase were performed for all samples. This cDNA was used as template for subsequent PCR with various primer pairs (Supplementary Materials and Methods). PCR conditions were similar to PCR genotyping with reduced cycles to avoid amplicon saturation. Semi-quantitative analyses of unsaturated amplicons were measured using ImageJ software. Intensity of bands was calculated as an arbitrary value relative to actin, beta (Actb) expression level.

Chiu A.P., Tschida B.R., Sham T.T., Lo L.H., Moriarity B.S., Li X.X., Lo R.C., Hinton D.E., Rowlands D.K., Chan C.O., Kam-wah Mok D., Largaespada D.A., Warner N, & Keng V.W. (2019). HBx-K130M/V131I promotes liver cancer in transgenic mice via AKT/FOXO1 signaling pathway and arachidonic acid metabolism. Molecular cancer research : MCR, 17(7), 1582-1593.

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Single Cell RNA Extraction and cDNA Synthesis

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Total RNA was extracted from single cell suspensions of either sorted CD4⁺CD44^high T cells or PaLNs of individual mice using the RNeasy Micro Kit (Qiagen, Valencia, CA). The entire sample was subjected to cDNA synthesis using the SuperScript™ First-Strand Synthesis System for RT-PCR (Life Technologies). The cDNA was frozen at −80°C before use as the template for PCR or sequencing.

Marrero I., Aguilera C., Hamm D.E., Quinn A, & Kumar V. (2016). High-throughput sequencing reveals restricted TCR Vβ usage and public TCRβ clonotypes among pancreatic lymph node memory CD4+ T cells and their involvement in autoimmune diabetes. Molecular immunology, 74, 82-95.

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